Calpain 3 (C3) is the muscle specific member of the calcium dependent protease family, mutations in which result in a form of muscular dystrophy called limb girdle muscular dystrophy type 2A (LGMD2A). In the previous funding period for this grant, we made good progress in understanding the biological function of C3. We showed that C3 is important for proper sarcomere structure, in vitro and in vivo. Based on our findings, we hypothesize that during normal development and muscle adaptation, C3, in conjunction with the ubiquitin/proteasome-system, displaces and removes myofibrillar proteins, and allows for the subsequent replacement by new proteins. Once sarcomeres are fully assembled they form such a dense compact entity that their subunits are protected from proteolytic action. C3's placement in the sarcomere, due to its interaction with titin, allows it accessibility to myofibrillar proteins that are to be turned over following damage or remodeling. We further showed that pathogenic LGMD2A mutations that do not affect C3s proteolytic capabilities can affect its ability to bind titin, and that titin serves as an important anchor for C3. Finally, we have shown that there is a generalized loss of protein turnover in C3KO muscles, leading to protein aggregates and a cellular stress response. The above observations have elucidated the biological function of C3 but have left unanswered questions. How does loss of C3's normal activity result in muscle disease? How does loss of anchorage to titin affect C3's normal cellular role? In our preliminary data, we show that C3 has cellular substrates that fall into two major categories that include myofibrillar proteins and mitochondrial proteins. This finding suggests that in addition to its role in myofibrillar protein turnover, C3 might be important in mitochondrial protein turnover. This hypothesis is supported by our preliminary data showing abnormal mitochondrial morphology and oxidative stress in C3 knock out mice. In this funding period, we will investigate the following aims: Aim 1: To characterize the biochemical consequences of disrupted calpain 3-titin interactions on calpain 3 activity and substrate accessibility. Aim 2: To characterize the biological consequences of disrupted calpain 3-titin interactions on muscle morphology and function. Aim 3: To characterize the mitochondrial defect and features of oxidative stress in calpain 3 KO muscles.